Activation of the immune system of vertebrates is an important mechanism for protecting animals against pathogens and malignant tumors. The immune system consists of many interacting components including the humoral and cellular branches. Humoral immunity involves antibodies that directly bind to antigens. Antibody molecules as the effectors of humoral immunity are secreted by B lymphocytes. Cellular immunity involves specialized cytotoxic T lymphocytes (CTLs) which recognize and kill other cells which produce non-self antigens. CTLs respond to degraded peptide fragments that appear on the surface of the target cell bound to MHC (major histocompatibility complex) class I molecules. It is understood that proteins produced within the cell are continually degraded to peptides as part of cellular metabolism. These fragments are bound to the MHC molecules and are transported to the cell surface. Thus the cellular immune system is constantly monitoring the spectra of proteins produced in all cells in the body and is poised to eliminate any cells producing non-self antigens.
Vaccination is the process of priming an animal for responding to an antigen. The antigen can be administered as purified protein, protein contained in killed/attenuated pathogens, or as a gene which then expresses the antigen in host cells (genetic immunization). The process involves T and B lymphocytes, other types of lymphoid cells, as well as specialized antigen presenting cells (APCs) which can process the antigen and display it in a form which can activate the immune system. Current modes for the administration of vaccines has focused on invasive procedures including needle injections, scarification, and gene gun-mediated penetration. Inoculation of vaccines in an invasive mode requires equipment and personnel with special medical training, and is usually associated with discomfort and potential hazards (bleeding, infection).
The efficacy of a vaccine is measured by the extent of protection against a later challenge by a tumor or a pathogen. Effective vaccines are immunogens that can induce high titer and long-lasting protective immunity for targeted intervention against diseases after a minimum number of inoculations. For example, genetic immunization is an approach to elicit immune responses against specific proteins by expressing genes encoding the proteins in an animal's own cells. The substantial antigen amplification and immune stimulation resulting from prolonged antigen presentation in vivo can induce a solid immunity against the antigen. Genetic immunization simplifies the vaccination protocol to produce immune responses against particular proteins because the often difficult steps of protein purification and combination with adjuvant, both routinely required for vaccine development, are eliminated. Since genetic immunization does not require the isolation of proteins, it is especially valuable for proteins that may lose conformational epitopes when purified biochemically. Genetic vaccines may also be delivered in combination without eliciting interference or affecting efficacy (Tang et al., 1992; Barry et al., 1995), which may simplify the vaccination scheme against multiple antigens.
Although topical application of protein-based vaccines in conjunction with cholera toxin may also immunize animals in a non-invasive mode (Glenn et al., 1998), skin-targeted non-invasive genetic vaccines activate the immune system via a different mechanism than protein-based vaccines. These two vaccination modalities may complement each other as they may induce different immune profiles. Although U.S. Pat. No. 3,837,340 relates to a method for vaccinating animals by contacting skin with dried viruses, the viruses that are employed therein are not genetic vectors capable of expressing transgenes or heterologous or exogenous nucleic acid molecules. In addition, the immunogen may be protein in the viral coat, instead of protein produced from recombinant DNA or expression of exogenous genes in the animals' own cells, and ergo U.S. Pat. No. 3,837,340 is non-analogous to the present invention.
Vaccination using live bacteria has been studied, and often utilizes a live bacteria strain in which a mutation has been induced to knock out the lethal gene. However, this method requires extreme safety precautions to ensure that a further mutation does not occur that would allow the bacterium to return to virulence. A more reliable method is to utilize a weakened bacterium to express a protein to which the host can then produce antibodies against. Often, a bacterial vector is studied for oral administration of a vaccine; for example, Salmonella-based vaccines are being researched for oral administration to protect against HIV, Lyme disease, and Epstein-Barr virus.
In addition, baculovirus, yeast and tissue culture cells have also been studied for use in vaccines, Examples are shown in U.S. Pat. No. 6,287,759 where baculovirus is employed to produce a protein used in a vaccine against Hepatitis E; U.S. Pat. No. 6,290,962 wherein yeast is used as a vector to produce a Helicobacter polypeptide for use in a vaccine; and U.S. Pat. No. 6,254,873 wherein vertebrate tissue culture cells are used to propagate purified inactivated dengue virus for use in a vaccine. In all of these examples, the vectors were used to produce a protein of interest, after which the protein would then be used in the vaccine.
Additionally, it has now been demonstrated (as evidenced by the following examples) that it can be advantageous to utilize irradiated bacterial vectors that are non-replicative. Non-replicative vectors are by nature safer than live vectors because there is no danger of mutations causing the vector to return to virulence.
Furthermore, it has now also been demonstrated (as evidenced by the following examples) that it can be advantageous to utilize cell-free extracts, wherein the extracts are prepared by filtration of disrupted cells chosen from the group consisting of bacterium, fungus, cultured animal cells, and cultured plant cells, and wherein the cell comprises and expresses a nucleic acid molecule encoding the gene product. These cell-free extracts can be applied directly to the skin, and are by nature safer than the use of live vectors.
Vaccines are often augmented through the use of adjuvants. Vaccine adjuvants are useful for improving an immune response obtained with any particular antigen in a vaccine composition. Adjuvants are used to increase the amount of antibody and effector T cells produced and to reduce the quantity of antigen and the frequency of injection. Although some antigens are administered in vaccines without an adjuvant, there are many antigens that lack sufficient immunogenicity to stimulate a useful immune response in the absence of an effective adjuvant. Adjuvants also improve the immune response from “self-sufficient” antigens, in that the immune response obtained can be increased or the amount of antigen administered can be reduced.
Heat shock proteins are a class of molecular chaperones which function by associating with cellular proteins and regulating their conformation. Heat shock proteins are located in all major cellular compartments and function as monomers, multimers, or are complexed with other cellular proteins. Heat shock proteins bind to steroid hormone receptors, repress transcription in the absence of the ligand, and provide the proper folding of the ligand-binding domain in the presence of the hormone. Specific heat shock proteins bind immunosuppressive drugs and can play a role in modulation of immune responses. In the present invention, it is demonstrated that the use of heat shock protein 27 can be used as a vaccine adjuvant to modulate immune responses.
The prior art of vaccination usually requires equipment, e.g., syringe needles or a gene gun, and special skill for the administration of vaccines. There is a great need and desire in the art for the inoculation of vaccines by personnel without medical training and equipment. A large number of diseases could potentially be immunized against through the development of non-invasive vaccination onto the skin (NIVS) because the procedure is simple, effective, economical, painless, and potentially safe. As a consequence, NIVS can boost vaccine coverages in developing countries where medical resources are in short supply, as well as in developed countries due to patient comfort. Infectious diseases caused by viruses, including AIDS and flu, by bacteria, including tetanus and TB, and by parasites, including malaria, and malignant tumors including a wide variety of cancer types may all be prevented or treated with skin-targeted non-invasive vaccines without requiring special equipment and medical personnel. The present invention addresses this longstanding need and desire in the art.
Additionally, the present invention also addresses the problems associated with new plastic surgery techniques involving the bacteria Clostridium (C) botulinum. In 2002, the Food and Drug Administration (FDA) approved the use of botulinum toxin A (Botox) for cosmetic treatment of glabellar lines. However, the current procedure requires multiple injections associated with a number of undesirable side effects.
The anaerobic, gram-positive bacterium Clostridium botulinum produces a potent polypeptide neurotoxin, botulinum toxin, which causes a neuroparalytic illness in humans and animals referred to as botulism.
Seven immunologically distinct botulinum neurotoxins have been characterized, these being respectively botulinum neurotoxin serotypes A, B, C.sub.1, D, B, F and G each of which is distinguished by neutralization with type-specific antibodies. The different serotypes of botulinum toxin vary in the animal species that they affect and in the severity and duration of the paralysis they evoke.
The neuroparalytic syndromes of tetanus and botulism are both caused by these neurotoxins produced by the bacteria. After binding to the presynaptic membrane of motoneurons, tetanus neurotoxin is internalized and transported retroaxonally to the spinal cord, where it blocks neurotransmitter release from spinal inhibitory interneurons. In contrast, the seven botulinum neurotoxins act at the periphery and inhibit acetylcholine release from peripheral cholinergic nerve terminals, inducing a flaccid paralysis due to intoxication of the neuromuscular junction. The clostridial neurotoxins responsible for tetanus and botulism are both metallo-proteases that enter nerve cells and block neurotransmitter release via zinc-dependent cleavage of protein components of the neuroexocytosis apparatus.
Besides the use of botulinum toxin A for cosmetic applications, botulinum toxins have been used in clinical settings for the treatment of neuromuscular disorders characterized by hyperactive skeletal muscles. Botulinum toxin type A has been previously approved by the U.S. Food and Drug Administration for the treatment of blepharospasm, strabismus and hemifacial spasm. Botulinum toxin type A is also being studied as a treatment for other neuro/muscular disorders including spasmodic dysphonia, dystonias in general, hyperhidrosis, and cerebal palsy.
Non-type A botulinum toxin serotypes apparently have a lower potency and/or a shorter duration of activity as compared to botulinum toxin type A. Clinical effects of peripheral intramuscular botulinum toxin type A are usually seen within one week of injection. The typical duration of symptomatic relief from a single intramuscular injection of botulinum toxin type A averages about three months.
The demonstration that topical application of a patch containing irradiated C. tetani cells could induce tetanus provides evidence and rationale in support of a novel protocol for the delivery of proteins capable of triggering beneficial pharmacological effects by topical application of irradiated bacterial cells containing the proteins using a patch. Topical application of a Botox patch will improve the degree of patient comfort and can eliminate some of the side effects associated with the contemporary needle-dependent method.